Effects of Extracellular Matrix on the Growth and Casein Gene Expression of Primary Mouse Mammary Tumor Cells in Vitro

This study describes a serum-free culture system and provides a tumor model to investigate the effects of extracellular matrices on the growth and β-casein gene expression of mouse mammary tumor epithelial cells (MMTCs) in vitro. Primary cultures of MMTCs derived from autochthonous mammary tumors in BALB/cfC3H x DBA/8 F, mice, FUKU cells, an established MMTC line, and COMMA-ID cells established from mouse mammary tissues were studied. A reconstituted basement membrane from the Engelbreth-Holm-Swarm tumor (Matrigel) allowed a 2.7-fold increase in cell number of 5-day primary MMTC cultures in serum-free, insulin-supplemented medium. FUKU and COMMA-ID cells in serum-free medium displayed a 13.6- and 11.5-fold increase in cell number, respectively, after 5 and 6 days in culture on Matrigel. In semisolid agar cultures, Matrigel or laminin was shown to promote colony-forming efficiency of FUKU cells when either of the matrices was mixed in the top agar layer. An increase of 4.4 or 2.1 times in colony-forming efficiency was detected when 20% (v/v) Matrigel or 112 Mg/ml of laminin were mixed in the agar layer compared with FUKU cells plated in plain agar. β-Casein mRNA was detectable by Northern blot assays in the primary mammary tumors. MMTCs in primary cultures grown on Matrigel in serum-free, insulin-supplemented medium for 4 days were inducible for β-Casein mRNA following the treatment with prolactin and hydrocortisone (FPRL) for 24 h. No β-Casein mRNA was detectable in the absence of FPRL. MMTCs in the primary cultures could also be induced for β-casein mRNA when they were cultivated on type I collagen gels for 4 days but not on laminin, type IV collagen, or plastic. However, the capacity to respond to FPRL was not lost in MMTCs cultured on laminin. When MMTCs were initially cultured on laminin for 4 days and then subcultured on Matrigel for another 4 days, they were inducible for β-Casein mRNA upon exposure to FPRL for 24 h. In contrast, no β-casein mRNA was found in MMTCs from the same tumors cultured on laminin for 8 days with the same treatment of hormones. These data demonstrate that cells from autochthonous mammary tumors, which are not dependent on estrogen for growth in vivo, are inducible in vitro for β-Casein mRNA by FPRL; and this hormonal response of MMCTs requires appropriate extracellular matrix.